Research Summary Human cytomegalovirus (CMV) is a ?-herpes virus that increases morbidity and mortality of immunocompromised individuals such as newborns, transplant recipients, AIDS patients, and the elderly. Common manifestations of CMV disease in some immunocompromised hosts include neuronal defects in infants and gastrointestinal disorders, pneumonia, CMV syndrome, and end-organ disease in transplant recipients. CMV is the leading cause of birth defects affecting 1-2.5% of newborns with approximately 40,000 new cases of CMV infection annually in the United States and range from 8-50% of solid organ transplant recipients (>145,000 globally) present with CMV-associated diseases. The anti-CMV FDA-approved drugs, ganciclovir, valganciclovir, foscarnet, cidofovir, and recently letermovir demonstrate limited efficacy and have severe drawbacks including poor oral bioavailability, dose-related toxicity, and promote the selection of drug resistant viral, respectively. CMV is a major health challenge that requires the development of a multi-faceted approach for the generation of effective and safe treatments to limit CMV proliferation. The objective of the current grant is to develop and utilize high throughput CMV infectivity assays to identify chemical inhibitors that target the diverse steps of CMV life cycle. Our central hypothesis is that compounds targeting the multiple steps of the virus life cycle can be developed into effective therapeutics that block infection, replication, and dissemination. The CMV life cycle (~96hrs) is a complex process requiring viral factors to manipulate cellular pathways to generate an infectious particle for dissemination by cell-free infections or cell-to-cell spread. To identify inhibitors of the different steps of the virus life cycle, we will use CMV variants that express two life- cycle stage-specific reporter proteins fused to yellow fluorescent protein (YFP): 1) the immediate early (IE)-2 gene product (AD169IE2-YFP), and 2) the early membrane protein unique short (US) 28 in a clinical-like strain (TB40/EUS28-YFP). In collaboration with Microbiotix, we plan to Aim 1: Perform a high content screening assay to identify inhibitors of the early steps of a CMV infection; Aim 2: Develop and apply high-throughput screening assays for the identification of inhibitors of the late stages phases of infection and viral spread; Aim 3: Validate and prioritize confirmed hits based on potency, specificity, and drug-like properties using a panel of orthogonal assays; and Aim 4: Define the mechanism of action for prioritized hit compounds. The development and utilization of several robust high-throughput assays will discover novel compounds that target multiple steps of the CMV life-cycle. The identified hit compounds have the potential to be developed into lead probes for CMV infection and dissemination. The prioritized inhibitors would limit CMV infection and dissemination in patients at risk for CMV-associated diseases.